Marc Bichara

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity.

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).

RECA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution.

RecA-mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesis.

In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF‐dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double‐stranded DNA sequences. Monomodified single‐stranded plasmids exhibit low survival in non‐SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10‐fold in a RecA‐dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA‐dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism – RecA‐mediated excision repair – in which RecA‐dependent pairing of the mono‐modified single‐stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.

Postreplication repair mechanisms in the presence of DNA adducts in Escherichia coli

During bacterial replication, DNA polymerases may encounter DNA lesions that block processive DNA synthesis. Uncoupling the replicative helicase from the stalled DNA polymerase results in the formation of single-stranded DNA (ssDNA) gaps, which are repaired by postreplication repair (PRR), a process that involves at least three mechanisms that collectively remove, circumvent or bypass lesions. RecA mediated excision repair (RAMER) and homologous recombination (HR) are strand-exchange mechanisms that appear to be the predominant strategies for gap repair in the absence of prolonged SOS induction. During RAMER, RecA mediates pairing of damaged ssDNA with an undamaged homologous duplex and subsequent exchange of strands between the damaged and undamaged DNA. Repair of the lesion occurs in the context of the strand-exchange product and is initiated by UvrABC excinuclease; the resulting patch is filled by DNA synthesis using the complementary strand of the homologous duplex as a template. HR uses a complementary strand of an undamaged homologous duplex as a transient template for DNA synthesis. HR requires the formation and resolution of Holliday junctions, and is a mechanism to circumvent the lesion; lesions persisting in one of the daughter DNA duplexes will normally be repaired prior to subsequent rounds of replication/cell division. Translesion DNA Synthesis (TLS) does not involve strand-exchange mechanisms; it is carried out by specialized DNA polymerases that are able to catalyze nucleotide incorporation opposite lesions that cannot be bypassed by high-fidelity replicative polymerases. Maximum levels of TLS occur during prolonged SOS induction generally associated with increased mutagenesis. RAMER, HR and TLS are alternative mechanisms for processing a common intermediate-the ssDNA gap containing a RecA nucleofilament. The actual pathway that is utilized will be strongly influenced by multiple factors, including the blocking/coding capacity of the lesion, the nature of the gene products that can be assembled at the ssDNA gap, the availability of a homologous partner for RAMER and HR, and protein:protein interactions and post-translational modifications that modulate the mutagenic activity of Pol-IV and Pol-V.

FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483–484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis

Arabidopsis RECG1 acts in mtDNA repair in the suppression of ectopic recombination and in the segregation of alternative mitotypes. The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.

Preferential post‐replication repair of DNA lesions situated on the leading strand of plasmids in Escherichia coli

In Escherichia coli, RecF‐dependent post‐replication repair (PRR) permits cells to tolerate the potentially lethal effects of blocking lesions at the replication fork. We have developed an in vivo experimental system to study the PRR mechanisms that allow blocked replication forks to be rescued by homologous sequences. We show that approximately 80% of the PRR events observed in SOS‐uninduced cells are generated by RecA‐mediated excision repair, a novel nucleotide excision repair‐ and RecA/RecF‐dependent mechanism, while 20% are generated by RecF‐dependent homologous recombination. Moreover, we show that in a wild‐type background, PRR is approximately an order of magnitude more efficient in processing DNA containing a blocked leading strand, as compared with a blocked lagging strand. This strand bias is abolished in cells that are deficient in nucleotide excision repair. These results are discussed in the context of recent models describing the mechanisms of replication past damaged templates.

Molecular Mechanisms of Mutagenesis Induced by Chemical Carcinogens and CIS-DDP

The conversion of DNA lesions into mutations is an active biochemical process. Due to the remarquable efficiency of the error free repair mechanisms only a very small number of DNA lesions will eventually be processed into a mutation. Under normal conditions, less than one percent of DNA lesions give rise to mutations. This makes the biochemical study of the mechanisms involved in mutagenesis very difficult. As a first molecular approach, the study of the mutationnal specificity of a given mutagen will provide important informations concerning the mechanisms that are involved. This is particularly true if the analysis of the mutational specificity can be performed in hosts having altered genotypes for repair and (or) mutagenesis. Bacteria are in this respect the organisms of choice, due to the large number of existing repair, replication and recombination mutants.

Substrate Specificity of the Formamidopyrimidine-DNA Glycosylase of E. Coli: Repair of the Imidazole Ring-Opened Form of N-Hydroxy-2-Amino-Fluorene-Guanine Adducts in DNA

A polynucleotide containing G-C8-AF residues was obtained by treatment of poly(dG-dC) with the carcinogen N-hydroxy-2-aminofluorene. The resulting product [3H]-AF-poly(dG-dC) was further incubated in 0.1 N NaOH for 24 hours at 37°C, which resulted in the conversion of 60% of the G-C8-AF residues to their imidazole ring-opened derivative (iro-G-C8-AF). This modified polynucleotide was used as substrate for the Fapy-DNA glycosylase of E. coli. H.P.L.C. analysis of the products of the reaction shows that the pure Fapy-DNA glycosylase excised the ring-opened derivative (iro-G-C8-AF). In contrast, the primary lesion (G-C8-AF) was not removed. These results show that the Fapy-DNA glycosylase of E. coli excises imidazole ring-opened purines which are modified at the C8 position. These observations suggest that the Fapy-DNA glycosylase may have a broad substrate specificity which includes all imidazole ring-opened purines modified at the N7 or C8 position in DNA.